Mitochondrial DNA Characterization of Five Species of Plebeia (Apidae
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Mitochondrial DNA characterization of five species of Plebeia (Apidae: Meliponini): RFLP and restriction maps Flávio de Oliveira Francisco, Daniela Silvestre, Maria Arias To cite this version: Flávio de Oliveira Francisco, Daniela Silvestre, Maria Arias. Mitochondrial DNA characterization of five species of Plebeia (Apidae: Meliponini): RFLP and restriction maps. Apidologie, Springer Verlag, 2001, 32 (4), pp.323-332. 10.1051/apido:2001132. hal-00891678 HAL Id: hal-00891678 https://hal.archives-ouvertes.fr/hal-00891678 Submitted on 1 Jan 2001 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie 32 (2001) 323–332 323 © INRA/DIB-AGIB/EDP Sciences, 2001 Original article Mitochondrial DNA characterization of five species of Plebeia (Apidae: Meliponini): RFLP and restriction maps Flávio de Oliveira FRANCISCO, Daniela SILVESTRE, Maria Cristina ARIAS* Departamento de Biologia, Instituto de Biociências, USP, São Paulo, SP, 05508-900, Brazil (Received 15 September 2000; revised 12 March 2001; accepted 30 March 2001) Abstract – The present work characterized the mitochondrial DNA (mtDNA) of five species of Ple- beia (Plebeia droryana, P. emerina, P. remota, P. saiqui and P. sp.) and generate a data set to be used in further populational, phylogenetic, and biogeographic studies. The mtDNA of each species was ana- lyzed using 17 restriction enzymes and restriction maps were built. A high level of interspecific vari- ability was found. The total size of the mtDNA was estimated to be 18500 bp. Through a combina- tion of PCR and examination of restriction fragment length polymorphism, the locations of 14 of the main mitochondrial genes were located on restriction maps. We verified a gene order identical to Apis mellifera. Plebeia / stingless bee / mtDNA / rflp / restriction map 1. INTRODUCTION (Kerr et al., 1996). The exclusively neotrop- ical genus Plebeia, morphologically one The tribe Meliponini (stingless bees) of the most primitive genera of this tribe exhibits a pantropical distribution and con- (Michener, 1990), is considered a post- tains a great diversity of species, including Gondwanan group that originated in the key species of several Brazilian ecosystems. region of southeastern Brazil (Camargo and Some species are essential for the pollination Wittmann, 1989). So far 30 species have of a great part of the Atlantic forest flora been described across a geographic range * Correspondence and reprints E-mail: [email protected] 324 F. de O. Francisco et al. from Mexico to Argentina (Michener, 2000). 2. MATERIALS AND METHODS Given the incomplete knowledge of the tax- onomy of the group, it is estimated that Ple- The Plebeia species studied were the fol- beia comprises about 40 species (Moure, lowing: Plebeia droryana (Friese), P. eme- unpublished data). Moreover, the biology rina (Friese), P. remota (Holmberg), of the described species is still poorly under- P. saiqui (Friese) and P. sp. (known as stood. Several systematic problems are com- “mirim-ponta-de-árvore”). The samples monly described (Camargo and Pedro, 1992) were collected from five nests (one for each and phylogenetic studies within the genus species) maintained at the Laboratório de are needed. Abelhas do Departamento de Ecologia do IB-USP. All the colonies were originally Mitochondrial DNA (mtDNA) is one of from Cunha, SP, except P. emerina, which the most widely used molecules in system- was from São Paulo, SP. atic, species characterization, population structure, and phylogenetic studies. In gen- 2.1. Restriction fragment length eral, animal mtDNA is a small, circular mol- polymorphism (RFLP) and Southern ecule, with a high evolutionary rate and a blot very conserved gene order and content (Gray, 1989). One of the characteristics of Two different procedures for total DNA mtDNA is a mix of conserved regions and extraction were applied. The TNE-protocol others showing a high substitution rate. (Sheppard and McPheron, 1991) worked Analysis by restriction fragment length poly- better for the species P. remota, P. saiqui, morphism (RFLP) technique allows the and P. sp. while the mini-TNE method study of this molecule as a whole, including (Arias and Sheppard, 1996) gave better both conserved and variable regions. results for the species P. droryana and P. emerina. All the extractions used 20 head- Detailed studies of bee mtDNA are recent less individuals each. and primarily restricted to the genus Apis. The DNA obtained by those extractions Apis mellifera L. mtDNA is approximately was digested overnight with the following 16300 base pairs (bp) long (Crozier and restriction enzymes: Bgl II, EcoR I, Hinc II, Crozier, 1993). Polymorphism of Apis Hind III, Pst I, Xba I, Xho I (GIBCO BRL) mtDNA has provided markers that have and BamH I, Bcl I, Cfo I, Cla I, EcoR V, been extremely important for the study of Hae III, Hpa I, Nde I, Pvu II and Sca I populations, hybridization, subspecies, and (Boehringer Mannheim). Each digestion species (Arias et al., 1990; Sheppard et al., used 5 U of enzyme and 5% of the total vol- 1991a; Sheppard et al., 1991b; Smith et al., ume of TNE extractions or 10% of the mini- 1991; Garnery et al., 1992; Lobo, 1995; TNE extractions. For double digestions, the Arias and Sheppard, 1996; Arias et al., 1996; DNA was digested with the first enzyme, Sheppard et al., 1996; Meixner et al., 2000). precipitated, and then digested with the sec- The great volume of data generated by the ond enzyme. analysis of this molecule in Apis encour- The digests were analyzed by elec- aged us to apply the same techniques to trophoresis in 0.8% agarose gels, stained related questions in other bees. As few with ethidium bromide, observed and pho- mtDNA studies have been reported for tographed under ultraviolet (UV) light. The Brazilian native bees (Costa, 1998), we DNA fragments were transferred to nylon report here the comparison and characteri- membranes (Amersham Pharmacia) using zation of mtDNA from five Plebeia species the Southern blot technique (Sambrook by restriction enzyme site mapping. et al., 1989) for further hybridization. The Plebeia mtDNA characterization 325 probe was derived from A. mellifera total 3. RESULTS AND DISCUSSION mtDNA, amplified through PCR (poly- merase chain reaction) in 12 fragments cov- The Plebeia species mtDNA was ana- ering the whole genome (Arias et al., 1998) lyzed by RFLP using 17 restriction enzymes. and labeled with digoxigenin using the Fragment sizes were calculated from South- DIG DNA Labeling and Detection Kit ern blot membranes (Fig. 1) and the total (Boehringer Mannheim). The labeling reac- length of Plebeia mtDNA was estimated to tion and membrane development were car- be about 18500 bp. This estimated size was ried out following the manufacturer’s pro- in agreement with the reported mtDNA tocol. Due to constraints of using genomes from other animals (Brown, 1985). heterologous probe, the hybridizations were No length variation among the species was conducted at 54 °C overnight. detected. Four of the 17 restriction enzymes (Cfo I, Hinc II, Hpa I and Pvu II) did not cut the 2.2. Polymerase chain reactions (PCR) mtDNA of any species. Based on the five species analyzed and the 13 remaining Total DNA from single individuals was enzymes, a total of 28 different restriction extracted using Chelex (Walsh et al., 1991), sites were generated. The enzymes BamH I and 5 µL of the supernatant were used as and Xba I cut the mtDNA only once and template for PCR reactions. Each reaction were species specific. Thirteen other restric- was set up with 5 µL of 10X PCR buffer, tion sites were also determined to be species 1.5 µL of each primer (20 mM), 5 µLof specific by absence or presence. A summary dNTPs (2 mM each), 2.5 U of Taq DNA of restriction site numbers generated per polymerase (Boehringer Mannheim) and enzyme for each species is presented in sterile water to achieve a final volume of Table II. 50 µL. The PCR amplifications consisted The total number of restriction sites per of an initial denaturation of 94 °C/5 min, species ranged from 23 (P. droryana) to 16 followed by 35 cycles of 94 °C/60 s for (P. remota), with 11 sites conserved among denaturing the DNA, 80 s at the appropriate the five species. Using double digestions temperature for annealing (Tab. I) and and the Southern blot technique, we could 64 °C/120 s for elongation. An additional construct initial restriction maps. However final extension step of 64 °C for 10 min was to further improve the resolution of such performed. The primers were derived from maps, 10 mitochondrial regions were ampli- A. mellifera (Hall and Smith, 1991; Arias fied for each species (Fig. 2). et al., 1998), Melipona bicolor Lepeletier Using A. mellifera mtDNA sequence as a (present work), and other organisms (Simon model for the position of the primers and et al., 1994). The PCR products were ana- respective amplified fragments, it was ver- lyzed in 0.8% agarose gels, stained with ified that approximately 13700 bp were ethidium bromide, visualized and pho- amplified, equivalent to about 75% of the tographed under UV light. Plebeia mitochondrial genome. The PCR-RFLP technique (Simon et al., The PCR-RFLP fragments were initially 1993) was also used. The PCR fragments used for the refinement of the restriction were digested with the same restriction maps. As an illustrative example, the PCR enzymes used for the total DNA in an product amplified by the primers AMB17 attempt to find sites very close to each other and AMB18 was approximately 1700 bp in and not detectable by Southern blot.